Comment[GEAAccession]	E-GEAD-741
MAGE-TAB Version	1.1
Investigation Title	5' and 3' single-cell RNA-seq of CD4+ cells and subpopulations
Experiment Description	To profile gene expression and enhancer activities at nucleotide resolution, we conducted 5'-end single-cell RNA-sequencing (5' single-cell RNA-seq). Using a novel 5' single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and non-coding RNAs in human CD4+ T cells, revealing immensely diverse cellular heterogeneity and differentiation trajectories.  *** Related raw data are to be made available through the controlled-access database JGA (study accession JGAS000689, https://humandbs.dbcls.jp/hum0350-v1). ***
Experimental Design	cell type comparison design
Experimental Factor Name	isolate	cell_subtype	replicate
Experimental Factor Type	isolate	cell_subtype	replicate
Person Last Name	Komatsu	Oguchi	Murakawa
Person First Name	Shuichiro	Akiko	Yasuhiro
Person Affiliation	RIKEN-IFOM Joint Laboratory for Cancer Genomics  Center for Integrative Medical Sciences, RIKEN		
Person Roles	submitter	submitter	submitter
Public Release Date	2024-05-14
Protocol Name	P-GEAD-2550	P-GEAD-2551	P-GEAD-2552	P-GEAD-2553	P-GEAD-2554	P-GEAD-2555
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol
Protocol Description	Blood samples were collected into vacuum tubes (Venoject II, Terumo, Cat#VP-H100K). CD4+ T cells were isolated by the immunomagnetic negative selection method using the MACSxpress Whole Blood CD4 T Cell Isolation Kit, human (Miltenyi Biotec). As for T cell subpopulations, The cells were stained by the antibody cocktail. The cocktail contained 50 microlitter of Brilliant Stain Buffer (BD Biosciences) supplemented with fluorochrome- conjugated antibodies: 5 microlitter CD25 (clone BC96, AF488, BioLegend), 20 microlitter LAG-3 (polyclonal, PE, R&D), 5 microlitter CXCR5 (CD185) (clone RF8B2, PerCP-Cy5.5, BD Biosciences), 0.75 microlitter CD3 (clone UCHT1, PE-Cy7, BioLegend), 2.5 microlitter CD196 (CCR6) (clone 11A9, APC, BD Biosciences), 1 microlitter CD45RA (clone HI100, APC-Cy7, BioLegend), 5 microlitter CD183 (clone 1C6/CXCR3, BV421, BD Biosciences), 1 microlitter CD4 (clone RPA-T4, BV510, BD Biosciences), 5 microlitter CD194 (CCR4) (clone L291H4, BV605, BioLegend), 7.5 microlitter CD197 (CCR7) (clone G043H7, BV711, BioLegend), and 0.5 microlitter CD8 (clone RPA-T8, PE-CF594, BD Biosciences). The fluorescence data were acquired by using a FACSAria IIu Cell Sorter (BD Biosciences) and the sorted cells were collected.	Human CD4+ T cells and FACS-sorted heterogenous populations were processed with Chromium Next GEM Single Cell 5' kits (10x Genomics). In brief, cell suspensions were loaded onto a Chromium single-cell controller (10x Genomics) to generate single-cell gel bead-in-emulsions (GEMs). Reverse transcription was performed in GEMs, generating cDNA tagged with a cell barcode and a unique molecular index (UMI), followed by barcoded cDNA amplification and library construction.	Library constructions were processed with Chromium Next GEM Single Cell 5' kit (10x Genomics). Libraries were sequenced on an Illumina Novaseq 6000 sequencing platform using paired-end, dual-index sequencing with 150 cycles for read 1 and 150 cycles for read 2.	Library constructions were processed with Chromium Next GEM Single Cell 5' kit (10x Genomics). Libraries were sequenced on an Illumina Novaseq 6000 sequencing platform using paired-end, dual-index sequencing with 150 cycles for read 1 and 150 cycles for read 2.	not applicable	Cell Ranger Software version 5.0.1 (10x Genomics) was used to process 5' Chromium scRNA-seq data (84). Reads were aligned to the Cell Ranger GRCh38 reference genome (refdata-gex-GRCh38-2020-A, 10x Genomics), and raw gene expression matrices were generated using the cellranger count function without the -include-introns option. CTSS files were generated by ReapTEC pipeline (https://github.com/MurakawaLab/ReapTEC).
SDRF File	E-GEAD-741.sdrf.txt
Comment[Number of channel]	single-channel
Comment[DBCLS]	The Data Access Committee of the Database Center for Life Science (DBCLS) approved that this personal data was made published according to the NBDC Guidelines for Human Data Sharing (https://humandbs.dbcls.jp/en/guidelines/data-sharing-guidelines) as the NBDC Research ID hum0350 and the application ID J-DS000641-001.
Comment[Array Design REF]	A-GEAD-11
Comment[AEExperimentType]	transcription profiling by array
Comment[BioProject]	PRJDB13816
Comment[Related study]	JGA:JGAS000689	NBDC:hum0350
Comment[Last Update Date]	2024-06-24